Symbiont integrity is therefore likely to be a prerequisite for the functioning of the cyanobacterial nitrogen fixing machinery.
The enslaved cyanobacteria may also provide energy-rich C-compounds or, as suggested for other symbiotic interactions, vitamin B12 production to it host [ ]. These hypotheses are yet to be investigated thoroughly. Phaeosomes are symbionts found in some representatives of the order Dinophysiales.
- Nitrogen-fixing bacteria in root nodules of leguminous plants.
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They exhibit morphological characteristics of Synechocystsis and Synechococcus cells and are located either extracellularly or intracellularly [ 94 ]. In the case of intracellular cells, the symbioses seem to be permanent and the benefit of the symbiosis to the host may be efficient nitrogen fixation. However, as in the case of P. Some filamentous cyanobacteria are known to interact with diatoms. Extracellular epibionts, endosymbionts and also symbionts positioned in the periplasmic space between the cell wall and cell membrane of the diatom are known to occur [ 58 , 98 ].
Electron microscopy scanning of such interactions has demonstrated a dual symbiotic nature of some symbionts. Richelia intracellularis has been observed to interact either as an epibiont with Chaetoceros spec. In these examples, nitrogen fixation for the benefit of the host has been demonstrated by the cultivation of the symbiont-diatom association in the absence of an external fixed nitrogen source. Nitrogen fixation is also suggested from morphological features such as the presence of heterocysts. At least in tropical environments, the production of B12 vitamins may also be a further benefit for the host [ ].
Some diatoms, including Climacodium frauenfeldianum and Rhopalodia gibba , are known to harbour permanent endosymbionts [ 96 , 97 , ]. As indicated by EM investigations of R. The endosymbionts, so-called spheroid bodies [ 96 ], are localised in the cytoplasm, and separated by a perialgal vacuole from the cytosol. Each spheroid body is surrounded by a double membrane. As additionally internal membranes are also visible, this morphotype is similar to that of cyanobacteria Figure 3b. Phylogenetic analysis groups these sequences together with free-living cyanobacteria of the genus Cyanothece Figure 2.
This robust grouping is also evidenced from phylogenetic analysis of a nitrogenase subunit gene, isolated from R. In phylogenetic reconstructions of both genes, the branch lengths separating free-living cyanobacteria and the cell inclusions of C. This is unlike the situation for plastids and extant cyanobacteria, which have an ancient phylogenetic relationship. Cyanothece sp. To protect the nitrogenase from oxygen tension, Cyanothece show a strong physiological periodicity, restricting nitrogen-fixation exclusively to the dark period of growth [ ]. During this period, the energy demand for N 2 fixation is sustained by large amounts of photosynthetically derived carbohydrates, which are stored as starch particles.
Nitrogen fixing activity of R. Intracellular localisation of the enzymatic activity has been undertaken by scanning for protein subunits of nitrogenase [ ]. Immunogold experiments have shown that the nitrogenase is localised within the diatom spheroid bodies, thereby confirming that the endosymbiont is responsible for the fixation of nitrogen. Furthermore, corresponding genes for the nitrogenase activity have also been isolated from purified spheroid bodies [ ].
Interestingly, spheroid body nitrogen fixation in R. This might be the result of several adaptations to the endosymbiotic lifestyle. Spheroid bodies lack a characteristic cyanobacterial fluorescence based on photosynthetic pigments, indicating that they have lost photosynthetic activity and that energy for nitrogen fixation is supplied by the host cell. The protection of the nitrogenase enzyme complex is accomplished through the spatial separation of the two pathways, with N 2 fixation in spheroid bodies and photosynthesis in the host plastid.
The loss of photosynthetic activity of spheroid bodies is also expected to lead to the loss of autonomy resulting in an obligate endosymbiosis. This hypothesis is consistent with the observation that R. Definitive evidence is still required to determine the exact nature of symbiotic interaction and whether the spheroid body of R. The ability to fix molecular nitrogen is restricted to selected bacterial species that express the nitrogenase enzyme complex. Nevertheless, various eukaryotic organisms have utilised this capacity by establishing symbiotic interactions with nitrogen fixing bacteria.
In these associations, fixed nitrogen is provided to the hosts, thereby enabling them to colonise environments where the supply of bound nitrogen is limited. In mutualistic symbioses, bacterial symbionts benefit from these associations, e. Symbioses for molecular nitrogen fixation can be found in many different habitats, with host organisms including all crown groups of eukaryotic life. Although all partnerships are based on the same enzymatic reaction, the diverse associations differ with respect to the physiological and morphological features that characterise the interconnection of partners.
Such features include the development of special host organs for optimal performance of bacterial symbionts, adaptations in host and symbiont metabolism, and the intracellular establishment of bacteria within the host. Close associations involving multiple adaptations and co-evolution between partners can result in permanent and obligate relationships, whereby the bacterial symbiont is stably integrated into the host system, and vertically transmitted across generations. These close interactions are mainly found in intracellular symbioses, where free-living bacteria reside within the cells of the host organism.
These are similar to organelles of eukaryotes, such as mitochondria and plastids, which both derived from symbiotic interactions and where continuous adaptation and co-evolution lead to a fusion of two distinct organisms [ 3 , 4 ]. In both cases, the metabolic capacity of the bacterial symbiont was the driving force for maintenance and evolutionary establishment, resulting in an inseparable merger of host and symbiont. The same basis of interaction applies for molecular nitrogen fixation, where eukaryotic hosts benefit from the unique metabolic capacity of special bacteria, leading to various symbiotic interactions with different specifications.
In particular, bacteria interacting with protists, like the spheroid bodies of R. The detailed study of this interaction will thus provide a great opportunity to understand the complex mechanisms underlying the evolution of obligate endosymbionts and organelles. Versammlung Deutscher Naturforscher und Arzte in Cassel. Martin W, Muller M: The hydrogen hypothesis for the first eukaryote.
J Phycol. Curr Biol. Theissen U, Martin W: The difference between organelles and endosymbionts. Biological nitrogen fixation. Fay P: Oxygen relations of nitrogen fixation in cyanobacteria. Microbiol Reviews. J Gen Micobiol. Yates MG: Effect of non-haem iron proteins and cytochrome C from Azotobacter upon the activity and oxygen sensitivity of Azobacter nitrogenase. FEBS Lett. Almon H, Bohme H: Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum.
Biochim Biophys Acta. Res Microbiol. J Bacteriol. Appleby CA: Leghemoglobin and Rhizobium respiration. Annu Rev Plant Physiol. Strain TM Appl Environ Microbiol. Mol Biol Evol. J Mol Evol. Hoffmeister M, Martin W: Interspecific evolution: microbial symbiosis, endosymbiosis and gene transfer. Environ Microbiol. Trends Ecol Evol. Mar Biol. Prog Mol Subcell Biol. Rai AN: Cyanobacteria-fungal symbioses: the cyanolichens.
Handbook of symbiotic cyanobacteria. Edited by: Rai AN. Marine and Freshwater Research. Pacific Science. Honigberg BM: Protozoa associated with termites and theit role in digestion.
Biology of termites. Arch Microbiol. Brune A: Symbionts aiding digestion. Encyclopedia of insects. Proc Biol Sci. Aksoy S: Tsetse — a haven for microorganisms. Parasitol Today. Applied and Environmental Microbiology. Termites: evolution, sociality, symbioses, ecology. Friedl T, Budel B: Photobionts.
Lichen Biology. Edited by: Nash TH. Honegger R: Functional aspects of the lichen symbiosis. Honegger R: The symbiotic phenotype of lichen-forming ascomyces. Edited by: Hock B. Plant and Soil. Cyanobacteria in Symbiosis. Tansley Review No. New Phytol. Parniske M: Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease?.
Curr Opin Plant Biol.
Brewin NJ: Development of the legume root nodule. Annu Rev Cell Biol. Stougaard J: Genetics and genomics of root symbiosis. Kistner C, Parniske M: Evolution of signal transduction in intracellular symbiosis. Trends Plant Sci. Phys Plant. Plant J. Mol Plant Microbe Interact. Bot Gaz.
Int Rev Cytol. Bashan Y, Holguin G, de-Bashan LE: Azospirillum-plant relationships: physiological; molecular; agricultural; and environmental advances. Can J Microbiol. Biology and environment: Proceedings of the Royal Irish Academy. Lindblad P, Bergman B: The cycad-cyanobacterial symbiosis. Meeks JC, Elhai J: Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states. Microbiol Mol Biol Rev. Am J Bot. Morphological aspects of the association.
Meeks JC: Symbiosis between nitrogen-fixing cyanobacteria and plants. Rasmussen U, Johansson C, Bergman B: Early communication in the Gunnera-Nostoc symbiosis: plant induced cell differentiation and protein synthesis in the cyanobacterium. Mol Plant-Microbe Interact. Margulis L: Origin of eukaryotic cells. Gray MW: Evolution of organellar genomes. Curr Opin Genet Dev.
Schnepf E, Schlegel I, Hepperle D: Petalomonas sphagnophila Euglenophyta and its endocytobiotic cyanobacteria : a unique form of symbiosis. Schnepf E: From prey via endosymbiont to plastid: comparative studies in dinoflagellates. Origin of plastids. Edited by: Lewis RA. Carpenter EJ: Marine cyanobacterial symbioses. Biology and environment. Proceedings of the Royal Irish Academy. Plant Syst Evol.
Floener L, Bothe H: Nitrogen fixation in Rhopalodia gibba ; a diatom containing blue-greenish inclusions symbiotically. Endocytobiology; Endosymbiosis and Cell Biology. Karl DM: Nutrient dynamics in the deep sea. Trends Microbiol. ATCC Plant Physiol. Molecular Plant-Microbe Interactions. J Ind Microbiol Biotechnol. Journal of Bacteriology. Download references. Correspondence to Christoph Kneip. CK and UGM conceived of this review and drafted the manuscript. CV and PL participated in preparing the final manuscript.
PL performed the phylogenetic analyses. All authors read and approved the final manuscript. This article is published under license to BioMed Central Ltd. Reprints and Permissions. Search all BMC articles Search. Abstract Background Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Results We have compared the morphological, physiological and molecular characteristics of nitrogen fixing symbiotic associations of bacteria and their diverse hosts.
Conclusion Our review emphasises that molecular nitrogen fixation, a driving force for interactions and co-evolution of different species, is a widespread phenomenon involving many different organisms and ecosystems. Background Historically, the phenomenon of symbiosis has been defined as a close and prolonged interaction between two different species [ 1 ].gatsbygroup.co.uk/battron-the-trojan-woman-vol1.php
Molecular nitrogen fixation and nitrogenase Most animals and fungi use nutrition to heterotrophically acquire nitrogen bound in biomolecules. Figure 1. Full size image. Figure 2. Table 1 Free-living and symbiotic nitrogen fixing bacteria Full size table. Results and Discussions Symbioses of nitrogen fixing bacteria with sponges, corals and insects invertebrates Marine sponges Porifera are evolutionary primordial invertebrates, which can harbour a variety of extra- and intracellular bacteria or bacterial communities [ 24 — 26 ].
Symbioses of nitrogen fixing bacteria with fungi: cyanolichens and symbionts of arbuscular mycorrhizal fungi In lichen symbioses, a fungal partner mycobiont is associated with an extracellular photobiont. Symbioses of nitrogen fixing bacteria with plants Interactions of bacteria with various groups of plants are the most common symbiotic association for nitrogen assimilation. Figure 3. Conclusion The ability to fix molecular nitrogen is restricted to selected bacterial species that express the nitrogenase enzyme complex.
References 1. Google Scholar 2. CAS Google Scholar 5. Google Scholar 6. Google Scholar 7. Google Scholar CAS Google Scholar PubMed Google Scholar Press Google Scholar Contact us Submission enquiries: Access here and click Contact Us General enquiries: info biomedcentral. To test the ability of the teosinte mucilage to support nitrogen fixation, we collected mucilage from several plants of teosinte and measured endogenous nitrogenase activity using ARA. Acetylene reduction was readily observed in teosinte mucilage Fig 4A , suggesting that production of mucilage that supports nitrogen fixation by an associated nitrogen-fixing microbiota may be an ancient trait of maize and potentially introgressed from Z.
To assess the mucilage characteristics that support nitrogen fixation, we tested the ability of 2 phylogenetically distinct nitrogen-fixing bacteria to reduce acetylene when inoculated in the mucilage collected from aerial roots of Sierra Mixe maize. Two nitrogen-fixing bacteria, H. Mucilage is also comprised of complex sugars that may be catabolized to provide free sugars—mainly arabinose, fucose, and galactose—capable of supporting bacterial growth and nitrogen fixation. To determine whether these properties of the mucilage are sufficient to support nitrogen fixation, we created an artificial medium mimicking these mucilage properties by using a low-N medium, solidified with 0.
Based on ARA, we can conclude that mucilage from Sierra Mixe maize harbors native diazotrophs and can also support the N 2 -fixing activity of the exogenously inoculated diazotrophs, H. However, these data did not demonstrate that the aerial roots had the capacity to take up and assimilate the fixed N.
To test whether atmospheric N 2 that was fixed by mucilage-associated diazotrophs could be transferred to and utilized by the Sierra Mixe maize, a more direct 15 N 2 gas—enrichment experiment was used. Aerial roots, along with their generated mucilage, inoculated with A. Mucilage was generated from aerial roots and inoculated with A. Mucilage alone and aerial roots alone were subjected to analysis by IRMS. Results revealed a significant enrichment of 15 N 2 in mucilage alone and aerial roots alone left y-axis.
Since the inoculated aerial roots may contain A. Results revealed a significant enrichment of 15 N 2 in these aerial roots, indicating that aerial roots are indeed the sites for transfer of fixed nitrogen to the plants right y-axis. The transfer of 15 N 2 from mucilage to the aerial root tissue and chlorophyll demonstrated the potential of this diazotrophic community to contribute to the nitrogen nutrition of the plant, but a major question of this study is whether the mucilage-associated diazotrophic microbiota served to transfer fixed nitrogen to fulfill, at least in part, the reduced nitrogen requirements of Sierra Mixe maize under field conditions.
The contribution of atmospheric nitrogen fixation to Sierra Mixe maize was first estimated in the field using natural abundance 15 N measurements [ 37 , 38 ]. This method relies on the relative abundance of the stable isotope 15 N in the atmosphere and soil, with 15 N abundance being more abundant in the soil than in the air.
In , samples of Sierra Mixe maize and reference plants from the Asteraceae and Ranunculaceae families with no known nitrogen-fixing members growing near each other were collected from each of 2 fields. In , , and , the methods from [ 38 , 39 ] were used in experiments in Sierra Mixe by sampling reference species of non-nitrogen-fixing plants growing near the Sierra Mixe maize plants and a conventional maize variety, Maiz Blanco Conasupo.
Values are given as mean and standard deviation. Statistical comparisons were made between the means of the reference plants, Maiz Blanco Conasupo, and Z. Different letters indicate statistically supported groups. Statistical comparisons were made between the reference plants mean and Z. Reference plants are listed in S3 Table. Several direct comparisons have indicated that both methods can give comparable but slightly different results [ 38 , 40 ].
The correlation between Ndfa and Ndiff was 0. Root and shoots exhibited significant differences in biomass, height, and stem diameter between control hybrids and local landraces S4 Table. Percent Ndfa and Ndiff were not calculated for reference dashes. We have demonstrated that the mucilage associated with the aerial roots of Sierra Mixe maize can support a complex diazotrophic microbiota enriched for homologs of genes encoding nitrogenase subunits that harbor active nitrogenase activity, and that nitrogen is transferred efficiently from the nitrogen-fixing bacteria to the host plant tissues.
Collectively, over several years and locations, the 15 N natural abundance and 15 N enrichment results of 2 selections of a Sierra Mixe indigenous maize landrace suggest that its nitrogen nutrition when grown in its native environment is partially fulfilled by fixation of atmospheric nitrogen. Nitrogen fixation is a particularly difficult phenotype to evaluate, as all the techniques available are prone to artifacts and can give different estimates for nitrogen fixation [ 42 , 43 ].
Original Research ARTICLE
This study also revealed a new and important function for aerial roots and the mucilage they produce besides preventing lodging or water uptake [ 30 ]. This role in nitrogen fixation is probably the most important one for aerial roots that do not reach the ground. It will be interesting to explore if aerial roots produced by other cereals such as sorghum can perform a similar function [ 44 ].
We cannot rule out that diazotrophic activity in other parts of Sierra Mixe maize may also contribute to the acquisition of reduced nitrogen from the atmosphere. The developmental timing of the appearance of fixed atmospheric N 2 in Sierra Mixe maize plants Tables 1 and 2 before the extensive development of aerial roots suggests that there may, indeed, be additional sites of nitrogen fixation.
However, we have not detected any significant nitrogenase activity outside of the aerial root mucilage. The genetic basis of the trait or the source of the microbial inoculum, which may be either environmental or seed-borne, are unresolved. The observation that a teosinte species Z. It will be important, in the future, to determine the genetic basis of the trait, the identity of associated microbial diazotrophs, and the mechanisms of microbial recruitment. This research, together with other published research [ 5 , 18 , 23 ], suggests new avenues for research into potentially novel mechanisms of biological N 2 fixation in maize.
This could have a significant impact on maize crop productivity and nitrogen use efficiency, particularly in regions of the world where agriculture is characterized by poor soil nutrition. Sierra Mixe maize seeds were obtained in Sierra Mixe region of Oaxaca, Mexico, from an open pollinated population. Maize line Hickory King was obtained from Victory Seeds accession SM1 is a uniform population based on kernel size, shape, and color, and SM2 is a heterogeneous population representing the landrace.
Bacteria were grown in liquid BSE medium. The rhizosphere and plant tissues that include stem, leaf, aerial roots, underground roots, and mucilage of Sierra Mixe maize were sampled during seasons , , and from Fields 1 and 2 in Sierra Mixe. Roots and stems were also dissected to remove epidermal tissues before extraction. For seed endophyte analysis, embryo and endosperm of Sierra Mixe, Hickory King, and B73 were withdrawn from the seeds by hand using a razor blade in a laminar flow cabinet and were collected in 1.
Roots were first separated and shaken to remove loosely adhering soil. All soils and plant material samples were used immediately for DNA extraction. Soil fertility analysis, which included physical parameters, soil reaction, and salinity, was performed in AgroLab Pachuma, Mexico. The number of nodes with aerial roots was monitored weekly greenhouse or after 14 weeks field. The total number of aerial roots was quantified after 14 weeks. The disappearance of leaf wax and appearance of trichomes were monitored weekly to determine the transition between juvenile stage and adult stage in Sierra Mixe and Hickory King maize.
For experiments in the greenhouse, seeds of Sierra Mixe of Fields 1 and 2 and Hickory King were surface sterilized and germinated as described previously. After 1 week, the seedlings are transplanted in liter pots filled with a mix of sand and perlite v:v and grown in a high-ceiling greenhouse at the Biotron facility University of Wisconsin, Madison, USA. Plants were watered twice a day for 2 minutes with half-strength of Hoagland solution.
For experiments in the field, 3 independent plots of 20 plants per genotype were planted, with 3 border rows B73 between each genotype. Sierra Mixe and Teosinte plants that were grown in Madison for the ARA were planted in the same field at the same time. This experiment was replicated in 3 different field plots. Gels were visualized by ethidium bromide staining under UV light in a gel documentation system. We extended the Caporaso approach [ 46 ] to a dual barcode scheme for each sample and replaced the Golay barcodes with a different set of Illumina-compatible barcodes that were designed to balance base composition and tolerate up to 4 sequencing errors in barcode sequences.
The barcodes used were designed to allow pooling of multiple samples within a single MiSeq run. Because of the low quality of the reverse mate pair, reads of the forward mate were used in the analysis. The reads were then preprocessed and analyzed using DADA2 [ 47 ]. Prescribed standard filtering parameters were used, such as PhiX contamination check and removal of reads with more than 2 errors or ambiguous bases or with an expected error greater than 2.
The clean reads were then collapsed into sequence variants and classified using RDP training set version The sequence variants that were classified as chloroplast or mitochondria were removed from further analyses. From samples with library size ranging from 84 to 20, reads mean of 4, , unique sequence variants were identified. Alpha and Beta diversity metrics were generated using the Phyloseq 3. Alpha diversity was calculated using Shannon and Simpson indices.
Additionally, a PCoA plot based upon Bray-Curtis dissimilarity matrix was used to visualize the differences in samples. The sequence variant table was used to generate a heat map following the variance-stabilizing transformation in DESeq2 [ 49 ]. Permanova tests were run using the adonis function from the vegan 3.
Illumina sequencing libraries from the same DNA extractions as above were made using an adaptation of the Nextera transposase-based library construction method with multiplex barcoding. Samples were then sequenced on the MiSeq and HiSeq instruments. Illumina sequences thus obtained were demultiplexed and trimmed using Trimmomatic ver 0. The clean reads were assigned taxonomy using Kaiju [ 52 ] with the nr database. To calculate the beta diversity of the samples, we used Phylosift ver 1.
Peptide sequences of the 6 core nif genes nifH , nifD , nifE , nifK , nifN , nifB and alternate nitrogenase anfG , vnfG from known diazotrophs as previously published [ 33 ] were retrieved from GenPept as a reference. A multiple-sequence alignment of these sequences was generated as a reference alignment using ClustalW2 [ 55 ]. The hits were then aligned against the multiple sequences alignment of reference using clustal-Omega [ 56 ] followed by generation of phylogenetic trees for every individual nif gene, using Fasttree2.
The reads were assigned as belonging to the nif genes if they were inside the clade of the reference sequences. Each read that had significant similarity to one of the core nif genes was further analyzed by phylogenetic analysis to confirm its assignment as one of the 6 core nif genes. For ARA with the mucilage, 2 ml of freshly collected mucilage from 1 or 2 aerial roots Sierra Mixe or several plants teosinte grown in the field were introduced in For ARA with added bacteria, A.
The Control tubes were prepared either without bacteria or with 5 ml of Fahraeus medium instead of mucilage. OD nm was measured for each tube after 72 hours. For both conditions, controls without acetylene were performed in parallel. For ARA with aerial roots, 1 aerial root without mucilage was introduced in each One ml of acetylene Airgas was injected into each vial.
Plants were then transferred to ml jars, and 50 ml of acetylene was injected in each jar. For ARA with underground roots, pieces of roots about 10 cm long were collected from plants grown in pots and introduced into The enrichment of mucilage in 15 N atom was achieved by removing 4 ml of headspace gas and replacing it with 4 ml of either 15 N 2 Sigma-Aldrich or 14 N 2 nitrogen gas directly into a vial containing 1. The samples were then freeze-dried and weighed. For measurement in collected mucilage, 2 ml of mucilage was introduced in a 15 ml tube. The probe robust oxygen mini probe, Pyroscience was introduced 8 mm deep in the mucilage and oxygen measurements performed until stabilization of the signal was observed.
Control corresponds to free-oxygen concentration in the liquid Fahraeus medium. One-point calibration was made in aerated water, as advised by the manufacturer. Mucilage was generated from each of these aerial roots by incubating them in 5 ml of water at room temperature for 48 hours. Mucilage, along with the aerial roots, was inoculated with A. After incubation of mucilage alone, or aerial roots alone, pheophytin extracted from these aerial roots was subjected to IRMS analysis. To obtain pheophytin, chlorophyll was extracted from aerial roots and converted to pheophytin by acid treatment, following as described [ 59 ].
In each of the and field seasons in Sierra Mixe, 90— individual maize samples depending on the year and reference plant samples, representing 8—10 species depending on the year of non-nitrogen-fixing plants, were analyzed. For the single time point in , 12 individual maize samples and 33 reference plant samples, representing 8 species of non-nitrogen-fixing plants, were analyzed. For 15 N natural abundance, the third-youngest leaf of Sierra Mixe maize or reference plants was collected from Field 3 and 4 from the second to the sixth month postplanting and analyzed for N-isotope composition.
Total organic nitrogen was determined by Kjeldahl digestion followed by steam distillation. Analysis for natural 15 N abundance was carried out as described by Bremer and van Kessel [ 38 ].
Biological nitrogen fixation in non-legume plants | Annals of Botany | Oxford Academic
In , 3 locations were chosen: Field 3, land that had not been planted to crops for over 10 years; Field 4, land that had maize for 1 year; and Field 5, land with continuous maize. A randomized complete block design trial was established at each site, with 5 replicates with 4 varieties. Each plot consisted of 6 matas surrounded by a common border of SM2 on all sides and a double border on outside rows. A mata is the traditional planting design in the Sierra Mixe region, similar to a hill plot in which multiple plants are seeded together.
Each mata was planted with 5 seeds and thinned to 3 seeds for 18 plants per plot. In , the same 3 locations were planted with same design and entries, except Field 3 consisted only of H and SM2 entries. In all 3 fields, 15 N was applied at a dose of 0. A solution was spread evenly over each plot using a garden watering can, such that the whole experimental area received an equal amount of enriched 15 N. Plants were covered with plastic bags at the time of application V5 to ensure that 15 N was not directly applied to the leaves and that the 15 N was uniformly available to all plants.
Soil samples were taken from a 0—60 cm depth in each plot, blended, and sent for analysis at UC Davis Soil lab. Means were calculated across locations. In , at V9 and V12, 1 mata 3 plants was sampled; and at Tassel, 4 matas 12 plants were sampled. In , a single sampling of 6 matas 18 plants was sampled at Tassel. For each sampling, plants were dug out to include all roots. Because of the high rainfall 2, mm concentrated in the growing season from June to October , roots were shallow for both reference and test varieties.
Each plant was photographed, and data were recorded for the number of plants, plant height, the total fresh weight of shoots, and roots and stem diameter. Whole plants were chopped, ground, subsampled, and dried in an oven to record total dry weight for shoots and roots. Well-blended subsamples were taken and shipped to Davis to measure Total N and 15 N. For Ndiff calculations, the area harvested was adjusted based on matas harvested at each time point. Data were analyzed using the R lme4 package. The heat map depicts the abundances of A 1, most abundant SVs and B 20 most abundant SVs in the dataset that were transformed using variance-stabilizing transformation in DESeq2.
A Number of nodes with aerial roots and B number of aerial roots observed on teosinte, Sierra Mixe maize, and Hickory King after 14 weeks. Different letters indicate statistically supported groups according to the Kruskal-Wallis test. A Oxygen measured at 3 depths in Fahraeus medium with black bars or without gray bars 0. B Effect of the different sugars present in the mucilage on the ability of H. From each location, 6 leaf samples were randomly sampled from Sierra Mixe maize plants and 6 leaf samples from each of 2 reference plants.
The third emergent leaf of each maize plant was sampled. Reference plants were selected from the most abundant weed species within each sample location, and from a plant family Asteraceae and Ranunculaceae that is neither actinorhizal nor leguminous nor has members known to associate with diazotrophic bacteria. Values are given as mean and s. Data are from a single sampling date May in Sierra Mixe maize, with 30 replicates analyzed for each sample reported. A Macroelements and soil characteristics. B Microelements for fields in This paper is dedicated to the life and memory of Cristobal Heitmann.
He died tragically while the manuscript was under review. We thank Vicente Vasquez and Maria del Refugio Vasquez for assistance in developing the program in Mexico; Carmen Ortega and Saulon Zamora for sample collection and field trial assistance; Shawn Kaeppler, Natalia de Leon, and Jillian Foerster for field assistance; Nguyet Dao for microbial 15 N-fixation assays; Harry Read for processing 15 N 2 -enrichment samples; John Zhang for assistance with library construction; and Armando Garcia-Llanos for assistance in sample processing. We thank the Comisiriado of the Sierra Mixe, Mexico, for their support and access to community genetic resources.
Abstract Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Author summary Nitrogen is an essential nutrient for plants, and for many nonlegume crops, the requirement for nitrogen is primarily met by the use of inorganic fertilizers. Introduction Plants grow in close association with microbial communities that influence plant traits related to nutrient acquisition, plant development, plant defenses, and abiotic stress responses.
Results Sierra Mixe maize morphology and mucilage The Sierra Mixe maize varieties cultured locally—referred to as Rojo, Piedra Blanca, and Llano—share similar plant morphologies, growing to a height of over 5 meters and exhibiting extensive aerial root formation at each node. Download: PPT. Sierra Mixe maize diazotrophic microbiota The microbiota associated with the underground and aerial roots, stems, and aerial root mucilage of Sierra Mixe maize grown in Sierra Mixe was investigated by amplifying and sequencing of 16S rRNA genes and by shotgun metagenome sequencing.
Fig 3. DNA sequencing—based characterization of the microbiome of Sierra Mixe maize. Fig 4. Nitrogenase and N 2 fixation activity in mucilage produced by Sierra Mixe maize. Fig 5. Analysis of Sierra Mixe maize samples for 15 N 2 enrichment in mucilage, aerial roots, and pheophytin from aerial roots. Nitrogen fixation contributes to maize N nutrition The transfer of 15 N 2 from mucilage to the aerial root tissue and chlorophyll demonstrated the potential of this diazotrophic community to contribute to the nitrogen nutrition of the plant, but a major question of this study is whether the mucilage-associated diazotrophic microbiota served to transfer fixed nitrogen to fulfill, at least in part, the reduced nitrogen requirements of Sierra Mixe maize under field conditions.
Table 2. Discussion We have demonstrated that the mucilage associated with the aerial roots of Sierra Mixe maize can support a complex diazotrophic microbiota enriched for homologs of genes encoding nitrogenase subunits that harbor active nitrogenase activity, and that nitrogen is transferred efficiently from the nitrogen-fixing bacteria to the host plant tissues.
Materials and methods Plant material Sierra Mixe maize seeds were obtained in Sierra Mixe region of Oaxaca, Mexico, from an open pollinated population. Bacterial strains and media A. Sample collection The rhizosphere and plant tissues that include stem, leaf, aerial roots, underground roots, and mucilage of Sierra Mixe maize were sampled during seasons , , and from Fields 1 and 2 in Sierra Mixe. Plant phenotyping The number of nodes with aerial roots was monitored weekly greenhouse or after 14 weeks field.
Greenhouse and field experiments, Madison, USA For experiments in the greenhouse, seeds of Sierra Mixe of Fields 1 and 2 and Hickory King were surface sterilized and germinated as described previously. Metagenomic sequencing Illumina sequencing libraries from the same DNA extractions as above were made using an adaptation of the Nextera transposase-based library construction method with multiplex barcoding.
Nif gene search Peptide sequences of the 6 core nif genes nifH , nifD , nifE , nifK , nifN , nifB and alternate nitrogenase anfG , vnfG from known diazotrophs as previously published [ 33 ] were retrieved from GenPept as a reference. Mucilage 15 N 2 assimilation The enrichment of mucilage in 15 N atom was achieved by removing 4 ml of headspace gas and replacing it with 4 ml of either 15 N 2 Sigma-Aldrich or 14 N 2 nitrogen gas directly into a vial containing 1. Measurement of free-oxygen concentration For measurement in collected mucilage, 2 ml of mucilage was introduced in a 15 ml tube.
Field data analyses Data were analyzed using the R lme4 package. Supporting information. S1 Fig. Box plot indicating the alpha diversity as calculated by Phyloseq using A Simpson index and B Shannon index. S2 Fig. Taxonomic distribution at the family level of the 25 most abundant bacterial families in A rRNA gene libraries and B whole-genome shotgun libraries. S3 Fig. Heat map showing the hierarchal complete linkage clustering of samples. S4 Fig. Nitrogenase acetylene reduction activity in different organs of Sierra Mixe maize.
Related Nitrogen Fixation in Bacteria and Higher Plants
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